Project Summary Site-Specific Incorporation of Phosphorylation Modifications on the SNAP-Tide High Density Peptide Array PIs: Mary S. Ozers and Christopher L. Warren Post-translational modifications (PTMs) increase the functional complexity and regulation of proteins, and dysregulation of PTMs are implicated in disease, including cancer. Almost all technologies used to detect and study PTMs depend on antibodies, tools that are as unreliable as they are ubiquitous. Antibody validation from manufacturers is highly variable, and antibodies, especially those directed against PTMs, suffer from off-target recognition. Our newly developed SNAP-Tide array (Specificity and Affinity for PepTides) is a high density, customizable, and high throughput peptide microarray platform technology, which can display up to one million unique peptides encompassing the entire human proteome on a single glass slide. This 100-fold increase in peptide density is possible because of our innovative synthesis process, in which peptide coding sequences on a standard DNA microarray are converted into RNA-barcoded peptides in vitro, and the resulting peptides are addressed back to the array. Peptide arrays are already used to analyze PTM enzyme specificity because many PTM enzymes, such as those that confer phosphorylation, sumoylation, and arginine methylation, target short linear peptide sequences rather than three-dimensional protein conformations. However, high density peptide arrays with PTMs already incorporated are not yet commercially available. In this proposal, we will innovate upon the SNAP-Tide platform to create the first commercially available modified high-density peptide array, the PhosphoSer SNAP-Tide array, which will display peptides containing phosphoserine (pSer), one of the most common PTMs in the cell. Specifically, we will: 1) Create a PhosphoSer SNAP-Tide array of the whole proteome, with single or multiple phosphoserines (pSer) per peptide using an orthogonal translation system (OTS) which allows site-specific incorporation of a PTM during translation; 2) Validate the presence and localization of pSer peptides on the array using biochemical techniques, including mass spectrometry, fluorescent labeling, antibody detection, and phosphatase treatment (Aim 2A), and compare to an existing peptide array modified enzymatically (Aim 2B); and 3) Demonstrate the utility of the PhosphoSer SNAP-Tide array by mapping the epitope of antibodies against estrogen receptor-? at key phosphorylation sites known to play a role in breast cancer (Aim 3A), and by examining the role of Pin1-catalyzed peptide bond isomerization in antibody recognition of pSer (Aim 3B). This technology allows the site-specific incorporation of both phosphorylated and non-phosphorylated serine residues at distinct positions within a million peptide sequences on a single array, something that cannot be achieved enzymatically. The PhosphoSer SNAP-Tide array could be employed by manufacturers and end-users to check antibody affinity (sensitivity), off-target binding (specificity), and lot-to-lot variation. Beyond antibody characterization, the PhosphoSer SNAP-Tide technology is anticipated to have a major impact on drug and antigen development, protein inhibitors, biomarker discovery, and diagnostic tools due to its unparalleled customization and affordability.